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Abstract

We present a prism-based spectrometer integrated into a multifocal, multiphoton microscope. The multifocal configuration facilitates interrogation of samples under different excitation conditions. Notably, the image plane of the microscope and the image plane of the spectrometer are coincident eliminating the need for an intermediate image plane containing an entrance slit. An EM-CCD detector provides sufficient gain for spectral interrogation of single-emitters. We employ this spectrometer to observe spectral shifts in the two-photon excitation fluorescence emission of single CdSe nanodots as a function of excitation polarization.

Figures (10)

A simplified representation of three types of spectrometers. (a) Specimen scanning spectrometers, where all colors are dispersed from a single point on the sample. (b) Wavelength scanning spectrometers, where one color at a time is selected from all points on the sample. (c) Prism-based spectrometer, in function similar to (a), but where the diffraction-limited spot size at the focus is the entrance slit.

Neon gas lamp calibration setup with slit formed of two razors, one of which is mounted on a translation stage. L1 located 2f from both the slit and second tube lens inside the IX-71. L1: 250 mm, L5: tube lens from Fig. 1 (provided for spatial reference), 170 mm.

Wide-field TPEF image of CdSe sample, collected with PMT. Pixel dwell time of 10 ms. Amplitude is in photon counts. The circled dot is examined with multiple polarizations in Figs. 9 and 10. Brighter spots than the one circled, but of the same lateral extent, are multiple emitters aggregated within the focal volume.

Spectra at orthogonal polarizations from single CdSe quantum dots. The dashed line corresponds to the spectrum obtained from unrotated excitation polarization. The dotted line corresponds to the spectrum obtained from excitation polarization rotated by 90°. Amplitudes are normalized to the maximum of the unrotated spectrum.

Time-series of TPEF signal from a single emitter, as collected by the PMT. Complete amplitude modulation, as seen around 120 s, is indicative of a single quantum dot. Though the dot appears “dead” after 150 s of exposure, a later spectral measurement did observe TPEF.